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Background Elevated levels of circulating non-esterified fatty acids (NEFA) are often observed during type 11 diabetes and have been proposed as a contributory factor for atherosclerosis. Experimental elevation of NEFA in healthy subjects impairs vascular function, but the impact of NEFA fatty acid composition is unclear. In vitro studies implicate a role for fatty acid-induced defects in insulin signalling, but these may be limited by unphysiological experimental exposures. Aim To examine the effect of varying NEFA concentration and fatty acid composition on vascular function, and explore the potential role of the endothelial insulin signalling pathway. Methods Two acute human studies elevated NEFA in healthy volunteers using oral fat loads of differing fatty acid composition together with a heparin infusion over 330 or 390min. Vascular function was assessed using flow-mediated dilatation (FMD), laser Doppler iontophoresis (LDI) or digital volume pulse (DVP). Regular venous blood samples were taken to assess lipid and insulin metabolism, as well as markers of endothelial function. In a subset of subjects, the notion that an infusion of insulin could restore vascular function was explored, this also served as a measure of whole-body insulin sensitivity (51). Extensive method development was undertaken to determine the impact of fatty acid incubations (single and mixtures) on proteins involved in the insulin signalling pathway in human aortic endothelial cells (HAEC). Results In the pilot study (10 males), an elevation of NEFA enriched in saturated fatty acids (5FA) versus mono unsaturated fatty acids (MUFA) was associated with an increase in a marker of arterial stiffness (DVPs,) and endothelial inflammation (sICAM). In the main study (30 males, 29 females), an elevation of 5FA-rich NEFA impaired FMD but a moderate substitution of 5FA for long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) reversed the impact, leading to a mean difference of 2.06 ± 0.29 % (P<0.001); LDI measures increased after both fat regimes (P~0.026) and there was no change in DVP indices. In the subset analysis, insulin infusion was not associated with an increase in FMD, although there was some indication that responses differed by gender. 51 was greater during the LC n-S PUFA regime compared with 5FA alone in males only (P=0.041). Preliminary in vitro work indicated that the MUFA, oleic acid increased the phosphorylation status of the enzyme which produces nitric oxide, a key vasodilator, however further method optimisation may be required. Conclusion These studies have shown that NEFA concentration and fatty acid composition can acutely modulate vascular function, and broadly support current advice to limit 5FA and increase LC n-3 PUFA consumption for prevention of heart disease. The large number of subjects included in the main study allowed us to show that both gender and genotype modulate the endothelial response to NEFA. A variety of mechanisms are likely to contribute to these findings and require further investigation in in vitro models which best replicate the physiological environment.